CN112011484B

CN112011484B - Screening and application of probiotic bacillus

Info

Publication number: CN112011484B
Application number: CN202010824181.5A
Authority: CN
Inventors: 石德时; 杨书敏; 罗吉; 周进; 肖运才; 李翔; 王喜亮; 李筱雯; 周祖涛
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-08-17
Filing date: 2020-08-17
Publication date: 2021-12-10
Anticipated expiration: 2040-08-17
Also published as: CN112011484A

Abstract

The invention belongs to the field of agricultural microorganism application, and particularly relates to screening and application of a probiotic bacillus strain. The invention relates to the screening and application of a prebiotic additive related to ruminant rumen digestion. The bacillus licheniformis strain with remarkable growth promoting effect on beef cattle is obtained by screening, and the application of the bacillus licheniformis strain in growth promotion of the feed additive for beef calves is verified through 16S rRNA gene identification, probiotic performance detection, safety evaluation and stress resistance identification. The Bacillus licheniformis strain (Bacillus paralicheniformis) SN-6 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020136. The strain has the advantages of high growth speed, strong stress resistance, safety and growth promotion, and can be used as a microecological additive for livestock and poultry feed.

Description

Screening and application of probiotic bacillus

Technical Field

The invention belongs to the field of agricultural microorganism application, and particularly relates to screening and application of a probiotic bacillus strain. The invention relates to the screening and application of a prebiotic additive related to ruminant rumen digestion. The bacillus licheniformis strain with obvious growth promoting effect on beef cattle is obtained through screening, and separation identification, probiotic performance detection, safety evaluation and stress resistance identification show that the bacillus licheniformis strain can be used as a feed additive for the growth promotion of the beef calves.

Background

A key step in the global carbon cycle is the hydrolysis of cellulose in the plant cell wall, which is the most abundant carbon source on land (Malhi 2003, David 2011). The reasonable utilization of cellulose resources can relieve pollution and solve global problems of global energy crisis, animal feed resource shortage and the like. The microorganism can hydrolyze cellulose to generate reducing sugar (cellobiose, glucose and the like) (Vu 2012), can improve the feed utilization rate, reduce the feed cost, improve the livestock productivity and has wide development prospect.

In animal husbandry and feed industry, in order to solve the problem of energy and feed shortage, people pay high attention to the separation and screening of microorganisms for producing cellulase and ligninase and the application of the cellulase and ligninase. Cellulolytic bacteria are known as "key species" of animal gut microbiome (suoshaofen 2019). Studies by McBee et al have shown that over 80% of cellulose digestion in ruminants depends on microorganisms in the rumen, and that feeding microorganisms can increase fiber utilization (McBee 1971). Sun et al report that after Bacillus subtilis feeding, digestion of cellulose in rumen of Holstein cows was improved, and daily gain of calves before weaning was significantly increased (Sun 2010).

The coarse feed in the beef cattle feed occupies a large proportion, but the coarse feed is rich in cellulose and lignin which are difficult to digest and utilize, and resource waste is caused. Buffalo and beef cattle belong to the same ruminant, and both have rumen as the main feed fermentation place, but the buffalo is more resistant to rough feeding than the beef cattle. It is reasonable to assume that: the rough feeding tolerance of buffalo is closely related to its unique gastrointestinal microorganisms.

At present, bacterial strains with strong activity of degrading cellulose, lignin and the like are not separated and screened from buffalo rumen, and reports of a bacillus licheniformis which has the capacity of degrading cellulose and lignin and has a remarkable growth promoting effect on beef cattle are not seen.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, separate and screen a safe probiotic strain with strong capability of degrading lignin and cellulose aiming at the technical barrier that the lignin and the cellulose in the coarse fodder are not easy to degrade, improve the utilization rate of the coarse fodder by beef cattle by using a microbial agent, improve the productivity and realize safety, high efficiency and environmental protection. Through 16S rDNA identification, the probiotic strain obtained by separation is Bacillus licheniformis (Bacillus Paralicheniformis), and the invention carries out relevant verification on the activity, safety and probiotics of the produced enzyme (cellulase, ligninase, amylase and protease) and the growth promoting effect of the probiotic strain as a beef cattle feed additive.

The technical scheme of the invention is as follows:

the invention separates and screens a candidate strain from the rumen of a buffalo resistant to rough feeding according to the screening target and the performance of special enzymes produced by probiotics as standards, the inventor identifies the obtained candidate strain as Bacillus licheniformis SN-6 and Bacillus paralicheniformis SN-6, and the candidate strain is delivered to China Center for Type Culture Collection (CCTCC) in Wuhan university in 2020, 05 and 21 months for preservation, the preservation number is CCTCC NO: m2020136.

Bacteriological characteristics of the strain Bacillus licheniformis (Bacillus paraleniformis) SN-6:

bacillus licheniformis (Bacillus parachutisti) SN-6 strain is gram-positive, is short Bacillus arranged singly, doubly or in a chain form and can form spores. On a flat plate taking sodium carboxymethylcellulose (CMC-Na) as a unique carbon source, a white, smooth and uniform colony with the periphery and the diameter of about 5mm is grown (see figure 1A), and a faint yellow hydrolysis ring is generated around the colony after Congo red staining (see figure 1B). The white lawn with the diameter of about 3cm can be grown by dibbling on a flat plate with sodium lignosulfonate as a unique carbon source (see figure 2). Growth on Potato Dextrose Agar (PDA) -guaiacol produces a reddish brown oxidation ring around the colony (see figure 3). The 16S rRNA gene is cloned and sequenced, and Blast comparison is carried out at NCBI, and the homology of the candidate strain 16S rDNA sequence of the invention and the bacillus licheniformis (MK517555.1) is the highest and is 99.65%.

The Bacillus licheniformis SN-6 strain has strong reproductive capacity, is in a lag phase after 0-4h, enters a logarithmic phase after 4h, and enters a stationary phase after 8 h. The Bacillus licheniformis SN-6 strain produces spores and has good stability. Tolerating artificial gastric juice and intestinal juice, and incubating in artificial gastric juice with pH of 3 for 3h to obtain a survival rate of 36.36%. The survival rate of the cultured cells in the neutral artificial intestinal juice is 54.29 percent after the cells are incubated for 4 hours. The strain is shown to have good tolerance, and is beneficial to being prepared into a microecological preparation and being applied to production as a feed additive.

Bacterial colonies of the strain SN-6 Bacillus licheniformis grow on a plate with sodium carboxymethylcellulose (CMC-Na) as a unique carbon source, and are stained with Congo red to form a light yellow hydrolysis ring around the bacterial colonies (see figure 1B), which indicates that the strain can hydrolyze cellulose and externally secrete cellulase. Growth on Potato Dextrose Agar (PDA) -guaiacol produced a reddish brown oxidation ring around the colony (see FIG. 3), suggesting that the strain produces laccase. Growing on LB plate containing 1% soluble starch, staining with gram iodine solution, forming white fading circle around colony, showing that the strain produces amylase (see figure 5), and can improve digestion utilization rate of energy substance starch.

The strain of Bacillus licheniformis SN-6 has obvious bacteriostatic activity on staphylococcus aureus and Escherichia coli K99 (see figure 7). The compound preparation is sensitive to common veterinary drugs such as penicillin, cephalexin, norfloxacin, tetracycline, vancomycin, chloramphenicol, furazolidone and compound neonomine, and has moderate sensitivity to cefuroxime; shows drug resistance to oxacillin. The virulence genes were detected by PCR and the strain was found to be free of relevant virulence genes (see FIG. 9). The mouse feeding test shows that the mice in the test group are not abnormal compared with the blank group. The strain is proved to have good safety.

The applicant prepares the SN-6 bacterial strain into a microecological preparation by a liquid fermentation mode, the microecological preparation is used as a feed additive, beef cattle feeding tests are carried out, the growth promoting effect on beef calves is obvious, the expected effect is achieved, and therefore the task of the invention is completed.

The bacillus licheniformis of the invention has the following advantages:

(1) the bacillus licheniformis SN-6 strain separated from the rumen of the coarse-feeding-resistant buffalo has stronger capability of degrading coarse fibers and obvious effect of improving the daily ration utilization rate of the beef cattle.

(2) The SN-6 strain has stronger reproductive capacity, produces spores and has strong stability; can resist artificial gastric juice and intestinal juice, and the survival rate is 36.36 percent after the artificial gastric juice with the pH value of 3 is incubated for 3 h; the artificial intestinal juice is incubated for 4 hours in a neutral state, the survival rate is 54.29%, and the preparation of the microecological preparation and the application of the microecological preparation as a feed additive in production are facilitated.

(3) The invention carries out safety panning on the SN-6 strain by using a molecular biology method and a mouse feeding test, and has higher safety.

(4) Animal experiments prove that the microecological preparation has a remarkable growth promoting effect on the beef calves, and the final purpose of the invention is achieved.

The more detailed technical scheme is shown in the content of 'concrete implementation scheme'.

Drawings

FIG. 1: the growth state of the separated and screened bacillus licheniformis strain SN-6 on a CMC-Na agar plate and the result of dyeing with congo red and decoloring with physiological saline are shown in the invention. Description of reference numerals: FIG. 1A in FIG. 1 is a graph showing the growth of the Bacillus licheniformis strain SN-6 isolated and selected by the present invention on CMC-Na agar plates. FIG. 1B in FIG. 1 is a graph showing the results of Congo red staining followed by destaining with physiological saline, showing the presence of a yellowish hydrolytic ring around the colony of SN-6.

FIG. 2: growth of Bacillus licheniformis SN-6 on a Lignin-derived sole carbon source plate.

FIG. 3: growth of Bacillus licheniformis SN-6 on PDA-guaiacol plates.

FIG. 4: is gram stain result (x 1000) of the separated and screened Bacillus licheniformis SN-6.

FIG. 5: is the detection result of the amylase produced by the strain SN-6.

FIG. 6: is the result of protease detection by the strain SN-6 of the invention.

FIG. 7: is the result of in vitro bacteriostasis of the supernatant of the SN-6 strain fermentation liquor. Description of reference numerals: FIG. 7, numeral 6 of the inhibition zone, is SN-6 strain isolated in the present invention. From left to right, the indicator strains used from top to bottom were in the order: escherichia coli O157, O139, K88, K99, Staphylococcus aureus, and Salmonella.

FIG. 8: is the virulence factor PCR detection result of the virulence gene positive strain bacillus cereus. Description of reference numerals: lanes in the figure: m is DL2000 DNA molecular weight standard; lane 1: nheA; lane 2: nheB; lane 3: nheC; lane 4: entFM; lane 5: and (5) negative control.

FIG. 9: is a glue pattern for detecting the corresponding virulence genes of the strain SN-6. Description of reference numerals: FIG. 9A: the invention relates to a PCR electrophoresis picture of a virulence gene nheA of a strain SN-6. Lanes in the figure: m is DL2000 DNA molecular weight standard; lane 1: the laboratory stores SN-20 strain; lane 2: the strain SN-6 of the invention; lane 3: the laboratory preserves the SN-4 strain: lane 4: the laboratory stores SN-3 strain; lane 5: a positive control strain; lane 6: and (5) negative control. FIG. 9B: the invention relates to a PCR electrophoresis picture of a virulence gene nheB of a strain SN-6. Lanes in the figure: m is DL2000 DNA molecular weight standard; lane 1: the laboratory stores SN-20 strain; lane 2: the strain SN-6 of the invention; lane 3: the laboratory preserves the SN-4 strain: lane 4: the laboratory stores SN-3 strain; lane 5: a positive control strain; lane 6: and (5) negative control. FIG. 9C: the invention relates to a PCR electrophoresis diagram of a virulence gene nheC of a strain SN-6. Lanes in the figure: m is DL2000 DNA molecular weight standard; lane 1: the laboratory stores SN-20 strain; lane 2: the strain SN-6 of the invention; lane 3: the laboratory preserves the SN-4 strain: lane 4: the laboratory stores SN-3 strain; lane 5: a positive control strain; lane 6: and (5) negative control. FIG. 9D: the strain SN-6 of the invention has an entFM (entFM) electrophoretogram of virulence genes. Lanes in the figure: m is DL2000 DNA molecular weight standard; lane 1: the laboratory stores SN-20 strain; lane 2: the strain SN-6 of the invention; lane 3: the laboratory preserves strain SN-4: lane 4: the laboratory stores SN-3 strain; lane 5: a positive control strain; lane 6: and (5) negative control.

FIG. 10: is the growth curve of the strain of the invention, i.e. the strain of the Bacillus licheniformis SN-6.

Detailed description of the preferred embodiments

Description of sequence listing:

sequence listing SEQ ID NO: 1 is the 16S rRNA gene partial sequence of the separated strain SN-6 of the invention.

Example 1: separation and identification of SN-6 strain of Bacillus licheniformis

First, strain isolation

The sample is taken from the healthy buffalo rumen provided with a rumen fistula by Hubei Tan cattle husbandry Limited company in Shayan county, Jingmen City, the rumen content is filtered by a warp cloth, and the filtrate is collected in an anaerobic bag and placed at 39 ℃ (vehicle-mounted small-sized incubator). 1mL of the collected sample was taken for 10^-1-10^-6Gradient dilution (100. mu.L of culture medium was added to 900. mu.L of sterilized water for gradient dilution), 10 were collected^-4、10^-5、10^-6Coating a CMC-Na unique carbon source plate with the three dilution culture solutions, placing the plate in an anaerobic box at 39 ℃ for culturing for 3 days, then picking a single colony, spotting the single colony on the CMC-Na unique carbon source plate, placing the plate in an anaerobic box at 39 ℃ for culturing for 3 days, then dyeing for 10-15min by using 0.1% Congo red dyeing solution, washing for 2 times by using 1.0mol/L NaCl solution, observing whether a light yellow hydrolysis ring is generated (Zhanger 2007), and picking a strain generating the hydrolysis ring for pure culture. Inoculating the pure culture strain on a sodium lignosulfonate sole carbon source plate and a PDA-guaiacol plate for sequential screening. The bacterial strain grows into white, smooth and regular colonies with the diameter of about 5mm on a flat plate with CMC-Na as a unique carbon source, as shown in figure 1A; a yellowish hydrolytic ring is generated around the colony after Congo red staining (see figure 1B); white colonies with a diameter of about 3cm were grown on a plate using sodium lignosulfonate as a sole carbon source, as shown in FIG. 2. Growth on PDA-guaiacol with a reddish brown oxidation ring around the colony (see figure 3); gram staining was positive, short rod-like, single, double or chain-like (see figure 4).

Secondly, identifying the strain species

On the basis of the identification, the 16S rRNA gene sequence detection is further carried out on the SN-6 strain of the bacillus licheniformis, and the species to which the SN-6 strain of the bacillus licheniformis belongs is confirmed and identified, and the specific steps are as follows:

firstly, extracting target strain genome:

(1) 1mL of the culture solution was centrifuged at 10000rpm for 30 seconds, and the supernatant was discarded as much as possible to collect the cells.

(2) 200. mu.L of buffer RB was added for resuspension, centrifuged at 10000rpm for 30s, and the supernatant was discarded.

(3) mu.L of lysozyme (20mg/mL in 10mM Tris-HCl, pH 8.0) was added thereto, mixed by inversion, and incubated at 37 ℃ for 30-60 min. Centrifuge at 12000rpm for 2min, discard the supernatant and resuspend in 180. mu.L of buffer RB by pipetting.

(4) Adding 20 μ L protease K (20mg/mL) solution, mixing, adding 200 μ L binding solution CB, immediately vortex, shaking, mixing, and standing at 70 deg.C for 10 min.

(5) After cooling, 100. mu.L of isopropanol was added and mixed well immediately by vortexing, whereupon a flocculent precipitate may appear.

(6) Adding the mixture (including the precipitate if any) in the previous step into an adsorption column AC, centrifuging at 13000rpm for 30-60s (placing the adsorption column into a collection tube), and pouring off the waste liquid in the collection tube.

(7) 500. mu.L of inhibitor-removing solution IR was added thereto, and the mixture was centrifuged at 12000rpm for 30 seconds, and the waste liquid was discarded.

(8) Add 700. mu.L of the rinse WB (with addition of absolute ethanol), centrifuge at 12000rpm for 30s, and discard the waste.

(9) Add 500. mu.L of the rinse WB (with addition of absolute ethanol), centrifuge at 12000rpm for 30s and discard the waste.

(10) And (4) putting the adsorption column AC back into an empty collection tube, centrifuging at 13000rpm for 2min, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction.

(11) Taking out the adsorption column AC, placing into a clean centrifuge tube, adding 50 μ L elution buffer EB (the elution buffer is preheated in water bath at 67-70 deg.C in advance) into the middle part of the adsorption membrane, standing at room temperature for 3-5min, and centrifuging at 12000rpm for 1 min. Adding the obtained solution into centrifugal adsorption column, standing at room temperature for 2min, and centrifuging at 12000rpm for 1 min.

(12) The DNA samples were stored at-20 ℃ until use.

(II) amplification of 16S rRNA Gene:

a bacterial universal primer is used for amplifying the 16S rRNA sequence of an isolated strain SN-6 strain of Bacillus licheniformis, the primer is synthesized by biological engineering (Shanghai) GmbH, and the DNA sequence of the primer is as follows:

forward primer 27F: 5' -AGAGAGTTTGATCCTGGCTCAG-3,

reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', respectively;

the PCR amplification system is shown in Table 1, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5min, pre-denaturation at 94 ℃ for 1min, denaturation at 50 ℃ for 30s, denaturation at 72 ℃ for 1.5min, 30 cycles, and elongation at 72 ℃ for 10 min. Taking the PCR amplification product to carry out electrophoresis detection on 0.8% agarose gel (containing ethidium bromide), wherein the size of the amplified fragment is consistent with the expectation and is about 1500 bp.

TABLE 1 PCR amplification System for Bacillus licheniformis SN-6 Strain

(III) constructing a bacterial evolutionary tree to determine the species:

extracting bacterial genome DNA, sending the bacterial genome DNA to a biological engineering (Shanghai) corporation Limited for sequencing, performing Blast on NCBI according to a sequencing result, constructing a bacterial phylogenetic tree by using MEGA7.0 software through an adjacency method (Neighbor-Joining), and displaying that a strain SN-6 is bacillus licheniformis according to an analysis result.

Example 2: probiotic property detection of Bacillus licheniformis SN-6 strain

First, enzyme production test

(I) detection of Amylase production Activity

Inoculating the strain of Bacillus licheniformis SN-6 on an LB plate containing 1% soluble starch, and culturing in a 37 deg.C incubator for 48 h; 2mL of iodine solution is added into the reacted starch culture medium, the mixture is slightly rotated until the iodine solution uniformly covers the flat plate, the mixture is kept still for 10min, the starch turns blue when meeting iodine, if amylase is produced, the starch around bacterial colonies is decomposed, a fading ring appears on a bluish-purple flat plate dyed by the iodine solution, the dyeing result is shown in figure 5, and the detection result of the amylase activity produced by the strain SN-6 of the bacillus licheniformis is shown in Table 2.

TABLE 2 detection results of amylase production by Bacillus licheniformis SN-6 strain

(II) detection of protease Activity

The strain of the Bacillus licheniformis SN-6 is spotted on a flat plate which takes milk as a unique carbon source, the flat plate is put into an incubator at 37 ℃ for culturing for 48 hours, if protease is produced, protein around bacterial colonies is decomposed, transparent rings appear on the flat plate, the result is shown in figure 6, and the detection result of the protease activity produced by the strain is shown in table 3.

TABLE 3 protease detection results of Bacillus licheniformis SN-6 strain

Second, bacteriostatic activity test of Bacillus licheniformis SN-6 strain

Escherichia coli O157, O139, K88 and K99, salmonella and staphylococcus aureus are used as indicator bacteria, supernatant of fermentation liquor of the strain is used as a bacteriostatic agent, and in-vitro bacteriostatic activity of the strain is detected, and the specific operation steps are as follows:

(1) preparation of suspensions of indicator bacteria (e.coli O157, O139, K88, K99, salmonella, staphylococcus aureus): streaking the indicator strain plate, and culturing at 36 ℃ for 20 h; picking single colony in LB liquid culture medium, placing at 37 deg.C, shaking and culturing for 16h, and recovering indicator bacteria. Adjusting the cultured indicator bacterium liquid to the bacteria number of 1.0 × 10⁷CFU/mL. (Note: dilution with 0.01mol/L PBS was required during dilution)

(2) Preparation and treatment of bacterial fermentation liquor: the strain plate is drawn, and cultured for 16h at 37 ℃. The isolated strain was cultured in 50mL of LB liquid medium at 37 ℃ and 200r/min for 24 hours to obtain a seed solution. The seed solution was inoculated into 100mL of LB liquid medium at an inoculum size of 1%, and cultured at 37 ℃ at 200r/min for 48 hours to give a fermentation broth. Centrifuging the prepared fermentation liquid for 5min under 10000r/min, and filtering the centrifuged supernatant with a 0.22 μm sterile filter for later use.

(3) And (3) bacteriostatic test: and dripping 100 mu L of the indicator bacterium suspension on an LB flat plate, uniformly coating a coating rod until no water drops are visible, taking the sterilized Oxford cup by using a sterile forceps, gently placing the sterilized Oxford cup on the surface of an LB solid culture medium, and uniformly placing 3 Oxford cups on each culture medium. Sucking 200 μ L of the supernatant of the strain fermentation broth by using a micropipette, and injecting into an Oxford cup which is placed stably (carefully adding, and not dripping on a culture medium outside the Oxford cup); the original glass plate lid was replaced with a sterile ceramic lid (to draw water evaporated from the medium during the subsequent experiments).

(4) Culturing: and (3) placing the culture medium added with the supernatant in a refrigerator at 4 ℃ for 8h, then placing the plate after complete diffusion treatment in a constant-temperature incubator at 37 ℃, and observing and recording test results after culturing for 16-24 h.

The test result shows that the strain has weak inhibition capability on pathogenic bacteria, wherein SN-6 has strong inhibition capability on K99 and staphylococcus aureus, and the result is shown in figure 7 and table 4.

TABLE 4 in vitro bacteriostatic test of the fermentation supernatants of the strains

Example 3: safety evaluation of Bacillus licheniformis SN-6 strain

First, drug sensitivity test

Selecting 15 drug sensitive paper sheets (purchased from Hangzhou microbial agent Co., Ltd.) of penicillins, cephalosporins, quinolones, aminoglycosides, tetracyclines, sulfonamides and the like for drug sensitive test. The test judgment standards were made with reference to the latest version of NCCLS standard provided by the World Health Organization (WHO) (version 2018). The method comprises the following specific steps:

(1) the bacterial liquid was inoculated into LB liquid medium in an amount of 1%, and cultured overnight at 37 ℃ with a shaker at 200 rpm/min.

(2) The turbidity was adjusted to 0.5 McLeod standard nephelometry against a black white paper background. If the concentration of the bacterial liquid is too high, the bacterial liquid can be diluted by physiological saline.

(3) Dipping bacteria liquid with a sterilized cotton swab, pushing the bacteria liquid against the tube wall to squeeze off redundant bacteria liquid, coating the bacteria liquid on an LB agar plate, rotating the plate for 60 degrees each time, finally coating the inner side edge of the plate for two circles, repeating the steps for several times, and ensuring the uniform coating.

(4) After the water on the plate is completely absorbed by the agar, the drug sensitive paper is clamped by a sterile forceps and pasted on the surface of the plate, and once the paper is pasted, the paper can not be picked up. 5 paper sheets are stuck on each flat plate, the distance between every two paper sheets is not less than 24mm, and the distance between the center of each paper sheet and the edge of the plate is not less than 15 mm.

(5) The plate is placed in a constant temperature incubator at 37 ℃ for 12-16h to observe the result.

The test results are shown in Table 5. The bacillus licheniformis SN-6 strain is sensitive to common veterinary drugs such as penicillin, cephalexin, norfloxacin, tetracycline, vancomycin, chloramphenicol, furazolidone and compound sulfamethoxazole, shows moderate sensitivity to cefuroxime and shows drug resistance to oxacillin.

TABLE 5 antibiotic susceptibility test results for Bacillus licheniformis SN-6 Strain

Note: s represents sensitivity, M represents medium sensitivity, and R represents drug resistance.

Second, detection test of virulence gene

The bacillus cereus containing non-hemolytic enterotoxin Nhe genes (nheA, nheB and nheC) and enterotoxin FM gene entFM is used as a positive control strain to detect related virulence genes of a target strain.

Respectively taking the genome of the prepared target strain as a template, and carrying out PCR amplification by using specific primers of various virulence genes, wherein the primers are synthesized by a biological engineering (Shanghai) corporation, and the PCR reaction condition is 94 ℃ for 3 min; 30s at 95 ℃, 30s at 58 ℃, 33s at 72 ℃ and 35 cycles; 72 ℃ for 10min (Rowan 2003). The virulence gene primer sequences and the expected PCR product sizes are shown in Table 6, and the virulence gene PCR amplification system is shown in Table 7.

TABLE 6 PCR amplification primer sequences for virulence genes

TABLE 7 virulence gene PCR amplification System

Note: negative control well DNA template was treated with 2. mu.L ddH₂And O is replaced.

The PCR product was electrophoresed on a 1.0% agarose gel (containing ethidium bromide), visualized in a gel imaging system and photographed. And (3) displaying an electrophoresis result: the positive strain amplifies 4 virulence genes nheA, nheB, nheC and entFM, the size of the amplified fragments is consistent with the expectation, and the result is shown in figure 8; no enterotoxin-related virulence genes were detected by strain SN-6, the results are shown in FIG. 9.

Safety feeding test for mice

Three-week-old Kunming mice (male and female halves) are selected to carry out the safe feeding test of the strain, and the test is carried outThe interval was 2 days pre-feeding acclimation period plus 2 weeks formal test period. Grouping according to body weight, wherein each group comprises 2 cages, and each cage comprises 5 cages; the test group mice are respectively gavaged with the target bacterial liquid (re-suspended and diluted by normal saline) of 200 mu L/mouse/day, and the bacterial quantity reaches 2-5 multiplied by 10⁸The blank control group was gavaged with an equal volume of physiological saline, weighed every 2 days, and the mental status and health status of the mice were observed and recorded every day.

And (4) analyzing results: the fur color and the mental state of the mice are observed, and the internal organs of the mice are observed through a autopsy, so that the mice fed by the target strain in a test group and the control group are normal compared with the blank group.

The results of the drug sensitivity test, the virulence gene detection, the mouse feeding test and the like prove that the bacillus licheniformis SN-6 is safe.

Example 4: stress resistance and growth characteristics of probiotic strain SN-6

Stress resistance test

(I) resistance to Artificial gastric juice test

(1) Preparing artificial gastric juice: preparing artificial gastric juice by referring to 'Chinese pharmacopoeia' 2015, accurately measuring 16.4mL of hydrochloric acid with the mass concentration of 100g/L, adding distilled water for dilution, adjusting the pH value to 3.0, then adding pepsin (the addition is calculated according to the mass concentration ratio of 1g/100 mL), fully dissolving, and filtering and sterilizing by using a microporous filter membrane with the pore diameter of 0.22 mu m to prepare the artificial gastric juice for later use;

(2) preparing a bacterial liquid: selecting a small amount of separated and preserved strains by using an aseptic inoculating loop, streaking the strains on an LB agar plate, culturing the strains in an incubator at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into an LB liquid culture medium, culturing the single colony at 37 ℃ until the logarithmic phase of the bacteria, counting the plates, and ensuring that the concentration of a bacterial liquid is 2.4 multiplied by 10⁹CFU/mL for standby.

(3) Inoculating the bacterial liquid into artificial gastric juice with pH value of 3.0 according to the inoculation amount of 1% of volume fraction, mixing uniformly, performing shake culture at 37 ℃ at 200r/min, sampling after 3h, and calculating the survival rate.

(II) test for resistance to Artificial intestinal juice

(1) Preparing artificial intestinal juice: preparing artificial intestinal juice by referring to 'Chinese pharmacopoeia' 2015, and weighing potassium dihydrogen phosphate KH₂PO₄3.4g, adding distilled water 2Dissolving 50mL, adjusting the pH value to 7.0 by using 0.4g/100mL NaOH solution, adding water to dilute to 500mL, then adding trypsin according to the mass concentration ratio of 1g/100mL, and filtering and sterilizing by using a microporous filter membrane with the pore diameter of 0.22 mu m after full dissolution to prepare artificial intestinal juice for later use;

(3) Inoculating the bacterial liquid into artificial intestinal juice with pH value of 7.0 according to the inoculation amount of 1% of the volume fraction, mixing uniformly, performing shake culture at 37 ℃ at 200r/min, sampling after 4h, and calculating the survival rate.

The survival rate of the strain resistant to artificial gastric juice/intestinal juice is calculated according to the following formula: survival (%) — number of viable/untreated viable/number of viable × 100; the results are shown in Table 8.

TABLE 8 detection results of artificial gastric juice/intestinal juice resistance of the strains

Second, measurement of growth Curve

(1) Preparing a seed solution: selecting a single colony, inoculating the single colony into 3mL LB liquid culture medium, and performing shaking culture at 37 ℃ for 24 hours to obtain a seed solution;

(2) preparing fermentation liquor: inoculating the seed solution into 100mL LB liquid culture medium according to the inoculation amount of 1%, and performing shaking culture at 37 ℃ for 24h to obtain a fermentation liquid;

(3) measurement of growth curves: inoculating the fermentation liquid into 50mL LB liquid culture medium at an inoculum size of 1%, taking out bacterial suspension at 0h, 2h, 4h, 6h, 8h, 12h, 16h, 20h, 24h, 28h and 32h respectively, and measuring OD₆₀₀Lower absorbance values. After 10-fold gradient dilution, the growth curve of each strain was measured by decantation, and each set of experiments was repeated three times. Using cultivation time as abscissa, OD₆₀₀Values are plotted on the ordinate, and growth curves of the strains are plotted.

Experiments show that the separated strain has strong reproductive capacity, is in a slow phase within 0-4h, continues to be 10h after entering a logarithmic growth phase within 4h, and enters a stable phase after 10 h. The strain has strong reproductive capacity and is beneficial to industrial large-scale fermentation production, and the growth curve is shown in figure 10.

Example 5: beef cattle feeding test

The probiotic strain SN-6 is prepared into a microecological preparation to feed animals (cattle) with more digested crude fiber, particularly beef calves, and aims to verify the growth promoting effect of the probiotic strain on the beef cattle.

First, test animal and group

And selecting Simmental meat calves with consistent genetic background as test animals, and pairing the calves one by one according to the weight before the test begins, wherein 9 pairs are obtained in total. Two cattle between each pair of pairs were randomly assigned to the placebo and test groups. The pens were housed in columns of 9 heads each, with a test period of 33 days. The strain SN-6 is prepared into bacterial powder through liquid fermentation, the number of viable bacteria reaches 66.7 hundred million CFU/g, the basic ration comprises corn straw silage, concentrate supplement, vinasse and wheat straw, and the concentrate supplement is purchased from Jingmen dragon valley feed limited company in Jingmen city of Hubei province.

Second, test procedure

The beef cattle feeding test is carried out by Liangyou Jinniu livestock-raising science and technology Limited in Hanqiao province of Shiqian town of Xiangyang, Hubei, the specific feeding time is from 22 days at 5 months in 2020 to 24 days at 6 months in 2020, and is 33 days in total. During the test period, the cattle only drink water and eat freely, the cattle are fed for 2 times (6: 30 in the morning and 16:00 in the afternoon), silage and concentrate supplement are mixed for feeding in the morning, wheat straw is fed in the afternoon, the feeding amount of the feed and the bacterial powder is increased and decreased according to the weight (taking beef cattle with the weight of 500 jin as an example, the complete feed is fed for 20 jin each day, the bacterial powder is added according to 7.5g of each cattle per day, and the total bacterial amount is 5.0 multiplied by 10¹⁰And/d/head), dissolving the bacterial powder in water and feeding the beef cattle.

Third, measuring the index

The influence of the bacillus licheniformis on the growth of beef cattle is detected through weight change, the first weighing is carried out on the fasting state before the probiotics are fed, the abdominal weighing is carried out once when the test is finished, and the average daily gain is calculated.

Fourth, result analysis

The results in table 9 show that, after 33 days of testing, the average daily gain of the blank control group was 1.24kg, the average daily gain of the test group was 1.37kg, and the average daily gain: test group > blank control group; compared with the blank control group, the average daily gain of the test group is remarkably different (P < 0.05).

TABLE 9 comparison of beef cattle growth performance

The relative weight gain of the blank group was 25.36% and the relative weight gain of the test group was 27.93% throughout the test period. The addition of the probiotic strains enables the relative daily gain of a test group to be increased by 10.82% compared with that of a blank group, and shows that the effect of adding the probiotic strains SN-6 of the invention in daily ration on the improvement of the growth performance of beef cattle is obvious.

Reference to the literature

1 MalhiYadvinder.Carbon in the atmosphere and terrestrial biosphere in the 21st century.[J].Philosophical Transactions of the Royal Society A:Mathematical,Physical and Engineering Sciences,2003,360(1801).

2 David B Wilson.Microbial diversity of cellulose hydrolysis[J].Current Opinion in Microbiology,2011,14(3).

3 Vu Van Hanh,KimKeun.Improvement of cellulase activity using error-prone rolling circle amplificationandsite-directedmutagenesis.[J].Journal of Microbiology and Biotechnology,2012,22(5).

4 suoshaofeng, study of gastrointestinal bacterial community composition and cellulolytic bacteria of mongolian horses [ D ]. university of inner mongolian agriculture, 2019.

5 Richard H.McBee.Significance of Intestinal Microflora in Herbivory[J].Annual Reviews Inc.,1971,2.

6 SUN P,WANG J Q,ZHANG H T.Effects of Bacillus subtilis natto on performance and immune function of preweaning calves[J].Journal of Dairy Science,2010；93(12):5851-5855.

7 Zhang super, plum brilliant guest, Zhang Lei, Zhang Qin, Weishiqing, cellulose-Congo red culture medium to identify the mechanism research of producing cellulase fungi [ J ] cellulose science and technology, 2007(02):39-44.

8 NEIL J.ROWAN G C C G.Production of Diarrheal Enterotoxins and Other Potential Virulence Factors by Veterinary Isolates of Bacillus Species Associated with Nongastrointestinal Infections[J].APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2003,69:2372-2376.

9 M.A.RATHER R S A J.Direct Detection of Bacillus cereus and its Enterotoxigenic Genes in Meat and Meat Products by Polymerase Chain Reaction[J].Journal of Advanced Veterinary Research,2011,1:99-104.

10 HENDRIKSEN B M H A.Detection of Enterotoxic Bacillus cereus and Bacillus thuringiensis Strains by PCR Analysis[J].APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2001,67:185-189.

11 NGAMWONGSATIT P,BUASRI W,PIANARIYANON P,et al.Broad distribution of enterotoxin genes(hblCDA,nheABC,cytK,and entFM)among Bacillus thuringiensis and Bacillus cereus as shown by novel primers[J].International Journal of Food Microbiology,2008,121(3):352-356.

12 national pharmacopoeia committee, pharmacopoeia of the people's republic of China [ M ]. Beijing, pharmaceutical science and technology publisher of China 2015.

Sequence listing

<110> university of agriculture in Huazhong

<120> screening and application of probiotic bacillus

<141> 2020-08-17

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1443

<212> DNA

<213> Bacillus licheniformis (Bacillus paralicheniformis)

<220>

<221> gene

<222> (1)..(1443)

<400> 1

agacgctgct atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg 60

gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac 120

cggggctaat accggatgct tgattgaacc gcatggttca attataaaag gtggctttta 180

gctaccactt acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240

aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300

ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360

gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaaa ctctgttgtt agggaagaac 420

aagtaccgtt cgaatagggc ggtaccttga cggtacctaa ccagaaagcc acggctaact 480

acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 540

aagcgcgcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 600

tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660

gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg 720

acgctgaggc gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780

taaacgatga gtgctaagtg ttagagggtt tccgcccttt agtgctgcag caaacgcatt 840

aagcactccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc 900

cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct 960

tgacatcctc tgacaaccct agagataggg cttccccttc gggggcagag tgacaggtgg 1020

tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080

cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac 1140

cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200

gctacaatgg gcagaacaaa gggcagcgaa gccgcgaggc taagccaatc ccacaaatct 1260

gttctcagtt cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc 1320

gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380

acgagagttt gtaacacccg aagtcggtga ggtaaccttt ggagccagcc gccgaagtga 1440

caa 1443

Claims

1. Separated and screened bacillus licheniformis (B)Bacillus paralicheniformis) SN-6 strain, preserved in China center for type culture Collection, with the preservation number of CCTCC NO: m2020136.

2. The use of a strain of Bacillus licheniformis SN-6 as claimed in claim 1 for the preparation of a micro-ecological additive for livestock and poultry feeding.